Ewing sarcoma is a bone and soft tissue tumor with a poor prognosis, particularly for patients with relapsed or metastatic disease, where overall survival is less than 30%. In the last 25 years since the establishment of the currently employed 5-drug chemotherapeutic regimen, there has been very little improvement in survival and no change in the therapy for patients with this disease. Therefore, there is a great need for new compounds and approaches that directly target the genes responsible for Ewing sarcoma cell survival. The goal of this proposal is to build a therapy centered on the suppression of the defining molecular feature of this tumor, the EWS-FLI1 transcription factor. Ewing sarcoma absolutely depends on continued expression of EWS-FLI1 for cell survival. This oncogenic transcription factor alters the expression of more than 500 genes to create the transcriptional program responsible for the continued proliferation of Ewing sarcoma cells, drug resistance and even metastasis. The challenge in targeting EWS-FLI1 is that the protein is a transcription factor and widely believed to be an undruggable target. In this proposal, we employ a bedside-to-bench and back again approach to develop a therapy that targets EWS-FLI1 using a compound called trabectedin as the backbone. In an early phase I study, a patient with Ewing sarcoma treated with Trabectedin achieved a complete response to this drug. Consistent with this result, we have previously shown that trabectedin interferes with the activity of the EWS-FLI1 transcription factor. Unfortunately, a follow-up phase II trial did not confirm that activity of th drug in Ewing sarcoma. In this proposal, we hypothesize that the failure of Ewing sarcoma patients to respond to the trabectedin in the phase II study was due to a poor therapeutic index of the drug that limited the exposure of trabectedin to levels that were not high enough to block EWS-FLI1. We believe that the drug associated toxicity that limited these serum levels was the collateral DNA damage that caused toxicity in normal cells as well as Ewing sarcoma cells. Therefore, in keeping with the NIH initiative of understanding extreme clinical responders, in this proposal, we will evaluate 82 analogs of trabectedin to identify compound(s) with an improved therapeutic index that will allow the blockade of EWS-FLI1 in patients. In the process, we will establish exactly how the drug works to block EWS-FLI1 activity and what the relative contribution of the drug associated DNA damage is to this activity. Finally, we will propose a novel combination therapy with improved EWS-FLI1 suppression that focuses the drug associated DNA damage specifically on Ewing sarcoma cells. Together, these results will provide the basis for the clinical translation of a trabectedin- based therapy centered on EWS-FLI1 blockade to improve patient survival.